Functional Study Of HaDFP2 Gene In Cotton Bollworm (Helicoverpa Armigera)

Prophenoloxidase activation reactions play an important role in insect innate immunity. PPO activation is a very complex system, A variety of molecules involved in the regulation of this response. We found a upregulated gene after immune stimulation by transcriptome sequencing of hemocyte in cotton bollworm (Helicoverpa armigera), which named as HaDFP2 (defense protein 2). The use of QRT-PCR,western blotting, protein functional assays in vitro and RNA interference method to explored the function of HaDFP2 gene in innate immunity of cotton bollworm, Helicoverpa armigera, the results show that the HaDFP2 gene may be involved in the regulation of prophenoloxidase activation reactions. Specific results are as follows:(1) Cloning and sequencing of HaDFP2 gene:Full-length of Ha-DFP2 for 425bp, the analysis found it has a complete open reading frame which can encode 123 amino acids. Predicted that the gene encodes a protein without signal peptide sequence, no transmembrane region, and with a molecular weight of 13.5 kDa, isoelectric point of 8.82.(2) Expression and purification of recombinant protein rHaDFP2 in prokaryotic express system and preparation of polyclonal antibodies:The use of expression vector pET-32a to induce and purify rHaDFP2 protein in E. coli, and obtained polyclonal antibodies.(3) Tissue-specific expression analysis:Use western blotting to detect the expression and distribution HaDFP2 protein in five kinds of tissues of hemocyte, plasma, fat body, midgut, and epidermis, the results showed that HaDFP2 protein only expression and distribution in plasma and hemocyte. This result indicates HaDFP2 protein is a secreted protein, upon expression by the hemocyte secreted into the hemolymph.(4) Expression levels of HaDFP2 gene in bollworm haemolymph by E.coli challenge:Analysis by QRT-PCR showed that E.coli challenge can significantly upregulate the expression of HaDFP2 gene in hemocytes, Meanwhile, analysis by western blotting showed HaDFP2 protein in bollworm hemocytes and plasma can also be upregulated by E.coli challenge. The results indicated that the gene is involved in the regulation of innate immune response of cotton bollworm, Helicoverpa armigera.(5) rHaDFP2 protein function tests in vitro:? rHaDFP2 can promote prophenoloxidase activation cascade in plasma, and promote the mechanism of beads in vivo. ? rHaDFP2 protein capable of binding with E. coli and S.aureus, and can contribute to both of them agglutination in vitro.(6) RNA interference test:After injection with dsHaDFP2 to knock down the expression level of HaDFP2 gene in hemocytes:the enzymic activity of phenoloxidase in plasma significantly decreased; and the ability of hemolymph clear E.coli and S.aureus significantly reduced.(7) 5'upstream region clone and promoter activity analysis of HaDFP2 gene:we amplified the 5 upstream region sequence of HaDFP2 gene for 3200bp, used the dual luciferase reporter system has been validated out 1005bp near 3'region sequence possess activity of promoter, and the promoter activity can be further enhanced by E. coli challenged. But there remains further experiments to identify the key sequence to the promoter activity.These results indicated that, HaDFP2 gene might play an important role in the innate immune response of cotton bollworm by mediating prophenoloxidase activation. The results also show HaDFP2 gene may be a new regulating factor of Prophenoloxidase cascade pathway, but the specific mechanism needs further study.